Earlier studies utilizing polyclonal antibodies and immunofluorescence (Goldenberg et al., Cancer Res. 36, 3455, 1976; Chakrabarty et al., J. Immunol. Methods, 43, 301, 1081; Taylor et al., Immun. Commun., 12, 315, 1983; Chakrabarty et al., Cancer Biochem. Biophys. 6, 249, 1983) showed that the cytosol fraction from the GW-39 tumor (a human/hamster xenograft) was a source of antigens expressed by a number of primary human colon tumors. Indirect immunofluorescence studies (Hilgers et al., Cancer Res. 32, 98, 1972) on human colon tumor cryosections using these polyclonal antisera and those of others (Arends et al., Biochim. Biophys. Acta 780, 1, 1985) have demonstrated moderate to bright immunofluorescence in many specimens obtained from human cancers of the bowel (Yeoman et al., Meth. in Cancer Res. 19, 233, 1982). Crossed immunoelectrophoretic analyses (Laurell, Scand. J. Clin. Lab. Invest. 29, 21, 1972) had shown that these polyclonal antisera were capable of recognizing more than 20 antigens (Chakrabarty et al., 1983). After extensive preabsorptions were performed with normal human and normal hamster tissues, only three antigens were detected. Using a quantitative filter-based radioimmunoassay (Chakrabarty et al., 1983), it was shown that elevated levels of colon antigen 3 (CA-3) expression could be measured in the extracts of primary human colon tumors but that negative or low levels were detected in the extracts of normal colon mucosa and the mucosa removed from nontumorous colon samples (Bara et al., Cancer Res. 44, 4040, 1984). Further biochemical characterization of the antigens recognized by these sera showed that they had molecular weights of 600-800 kilodaltons (Chakrabarty et al., 1983).
Inasmuch as quantitative data obtained with polyclonal antisera had indicated that immunoassays based upon reactivity with antigens of very high molecular weight could discriminate between samples of colon cancers, normal adjacent colon and normal colon specimens, the present inventors began experiments in which selected immunizations were done with antigens of high molecular weight (600-800 kilodaltons). The present invention has resulted from studies designed to produce monoclonal antibodies to human tumor antigens of high molecular weight and to detect their presence in peripheral blood specimens. The advantages of monoclonal antibodies over the polyclonal antibodies used in the previous studies are: (1) the high specificity of monoclonal antibodies (2) the potentially unlimited supply of antibody and (3) the absence of a need for absorptions to improve their specificity. The advantages of a serum based test over the assay of extracts prepared from tumor specimens are: (1) the ease of sample acquisition, (2) the ability to screen for cancer in patients at high risk for specific types of cancer and (3) the possibility of early detection in asymptomatic individuals and (4) the ability to subsequently follow therapy or reoccurrence of disease.
Antigen gp650 has a molecular weight of approximately 650 kD as determined by gel filtration on a calibrated S-300 Sephacryl column and has been shown by immunofluorescence to be localized within the cytoplasm of human colon tumor cells that express this antigen.
The following Table 1 presents a summary of the characteristics of antigen gp650. The antigenic determinant recognized by the monoclonal antibody is on this protein and is distinct from carcinoembryonic antigen (CEA) with regard to its immunoreactivity and its chromatographic behavior (Kessler et al., Cancer Res. 38, 1041, 1978).
In Table 2, the clinical specificity of the monoclonal antibody is shown. Elevated levels (Table 3) of gp650 were found in the sera of patients with gastrointestinal cancers, cancers of the breast, cancer of the lung, cancer of the tongue, fallopian cancer, lymphoma, hepatomas and multiple myelomas. Normal human serum and primary biliary cirrhosis sera did not have elevated levels of gp650.
TABLE 1 ______________________________________ Properties.sup.a of Human Cancer Antigen gp650 650 kilodalton molecular weight (native) 300 kilodalton molecular weight (denatured) Glycoprotein Cytosolic in origin and localization In serum of cancer patients Protein determinant Derived from high molecular weight fraction of GW-39 tumor cytosol (human/hamster xenograft) Destroyed by trypsin, pronase, HNO.sub.2 and 2-mercaptoethanol Immunologically and chromatographically distinct from CEA (carcinoembryonic antigen) Insensitive to treatment with chondroitinase ABC, endoglycosidase H, mixed endoglycosidases and DNase I ______________________________________ .sup.a Molecular weights were determined by gel filtration on a calibrate Sephacryl S300 column in the presence (denatured) and absence (native) of 8 M guanidinehydrochloride. Antigen gp650 was treated with proteases, endoglycosidases, nitrous acid, reducing agent, chondroitinase ABC and DNase I to determine its composition, sensitivities and insensitivities. The gel filtration characteristics of carcinoembryonic antigen were determined by ELISA assay using a commercial antiserum.
TABLE 2 ______________________________________ Clinical Specificity.sup.a of the Monoclonal Antibody Specific for Antigen gp650 Cancer Specimens ______________________________________ gastrointestinal 8/9 breast 4/4 hepatoma 3/3 multiple myeloma 2/2 lymphoma 1/2 lung 1/1 fallopian 1/1 tongue 1/1 uterine 0/1 prostate 0/1 esophagus 0/1 Non-cancer normal serum 0/3 primary biliary cirrhosis 0/1 ______________________________________ .sup.a Immunoreactivity was determined by a direct binding ELISA assay using antigen bound to plastic microtiter plates. Following incubation with primary antibody, a horse antimouse biotinylated second antibody was added followed by an avidinbiotinylated horseradish peroxidase complex. Immunoreactivity was measured by quantitating the amount of bound horseradish peroxidase with the addition of 2,2'-azinodi-(3-thylbenzthiazolinsulfonat(6)) diammonium salt (ABTS) and hydrogen peroxide followed by the development of color (Kelsey et al., Cancer Letters 12,295, 1981).
TABLE 3 ______________________________________ Quantitative Immunoreactivity.sup.a of Human Serum Specimens with a Monoclonal Antibody to Human Cancer Antigen gp650 Units.sup.b of Number of Immunoreactivity Specimens Samples Range.sup.c Mean ______________________________________ Cancers: hepatoma 3 14-75 39 lung 1 30 30 breast 4 10-45 30 fallopian 1 29 29 myeloma, multiple 2 8-32 20 colon 9 0-45 16 tongue 1 6 6 lymphoma 2 0-10 5 uterus 1 4 4 prostate 1 0 0 esophagus 1 0 0 Non-cancers: normal serum 3 2 2 primary biliary cirrhosis 1 2 2 ______________________________________ .sup.a Immunoreactivity was determined by the direct binding ELISA method described in the legend to Table 2. .sup.b The Units are expressed in ng of S300 Sephacryl purified gp650 per ul of serum. .sup.c Values are based upon a minimum of triplicate analyses.